The therapeutic benefits of increased gamma globin expression in the presence of hemoglobinopathies such as sickle disease are well documented. Unfortunately, the molecular control mechanisms which regulate the postpartum decrease in gamma globin levels are still not well understood. Recently it has been shown that certain unique DNA sequences have the ability to form triple helices with complementary third strands occasionally leading to alterations in the cognate gene expression. Triple helix formation, referred to as Hoogsteen base pairing, occurs between purine and pyrimidine nucleotides according to the following schemata, A-AT, T-AT, G-GC,C+-GC. The uniqueness of potential triplex regions in native DNA as well as the increased binding stability over double stranded (ds) DNA has led to the development of triple helix forming oligonucleotides as site specific controls of gene expression. Several studies have shown that the formation of DNA triple helices at specific enhancer sites results in decreased expression of select target genes. Our study focused on a 20 b.p. long purine rich region of DNA approximately 189 b.p. upstream from the gamma globin cap site. Based on prior evidence we believe this target region to have silencer activity which is activated by a repressor protein which binds in the region from -202 through -196 b.p. We have used specially designed triple helix forming oligonucleotides to serve as a site specific inhibitor of repressor protein binding in the -202 through -196 b.p. region of the gamma globin promoter. By inhibiting binding of the repressor protein we hoped to thereby increase the levels of gamma globin gene expression. A variety of in vitro and in vivo studies were used to assess the effect of triple helix formation in both the transformed K562 erythroleukemia cell line, and erythroid cells derived from progenitor cells in circulating mononuclear cells of normal individuals. We were able to successfully demonstrate in vitro triple helix formation within our target region in the gamma globin promoter. The in-vivo data is still being developed.